肝星状细胞与肝纤维化

肝纤维化(hepatic fibrosis)是指肝脏纤维结缔组织的过度沉积,是细胞外基质(extracellular matrix,ECM)合成和降解不平衡的结果,是各种慢性肝病向肝硬化发展所共有的病理改变和必经途径。而肝星状细胞(hepatic stellate cell,HSC)是ECM产生的主要细胞来源,它的激活、凋亡受到众多细胞因素及细胞内信号转导系统的调节,因此阐明肝星状细胞的生物学特性与功能及其在肝纤维化中的作用机制,对肝纤维化的预防和治疗都有     

肝星状细胞与肝纤维化

肝纤维化是一切慢性肝病的共同病理学基础 ,是形成肝硬化的必经病理阶段。肝纤维化的形成机理目前多数认为是致病因子造成肝细胞 (HC)损伤 ,引起肝枯否氏细胞 (KC)激活 ,分泌多种细胞因子 (CK) ,随同血小板、肝窦内皮细胞 (SEC)和肝细胞等分泌多种细胞因子 ,与某些化学递质共同作用于肝星状细胞 (HSC) ,使其激活 ,转化为肌成纤维细胞 (MFBLCs) ,通过旁分泌与自分泌作用 ,使 HSC增殖 ,合成大量的细胞外基质(ECM) ,ECM的分泌增加 ,降解减少 ,以致其在肝内大量沉积 ,肝纤维化逐渐形成 [1]。但肝纤维化与其它组织纤维化不同 ,很少有…     

莪术对肝星状细胞活化的影响

目的:观察莪术对肝星状细胞活化的影响。方法:莪术水煎液(0.3g/ml)按10ml/kg 大鼠体重给正常大鼠灌胃,每天2次,连续3天,第4天1次给予全日剂量,1h 后采血,离心分离血清,56℃灭活,制备药物血清。以正常鼠血清及秋水仙碱药物血清作为对照。将药物血清温育传一代的肝星状细胞,氚标胸腺嘧啶核苷(~3H-TdR)掺入法及噻唑蓝(MTT)比色法测定细胞增殖;酶联免疫吸附(ELISA)法测定培养上清Ⅰ型胶原含量,改良 Lowrv's 法测定细胞层总蛋白以校正细胞数;貂肺上皮细胞株-Mvllu 抑制 MTT 法测定培养上清转化生长因子β_1(TGF β_1)。结果:莪术药物血清和秋水仙碱药物血清对活化的肝星状细胞增殖均有显著抑制作用(P<0.01)。莪术的抑制作用强于秋水仙碱(P<0.01),二者都能抑制肝星状细胞生成Ⅰ型胶原(P<0.01),并且都能显著抑制肝星状细胞分泌 TGF β_1,两者作用比较,差异无显著性(P>0.05)。结论:莪术能抑制肝星状细胞增殖,减少胶原和 TGF β_1的生成,从而抑制肝星状细胞的活化。     

肝星状细胞的凋亡与肝纤维化的研究进展

<正>肝星状细胞(HSC)是肝脏的一种间质细胞,肝纤维化(HF)是肝脏对各种原因所致肝损伤的创伤愈合反应,由于损伤引起HSC激活及其转化为肌成纤维细胞(MF),从而大量合成各种细胞外基质(ECM)沉积于肝脏,最终导致HF。激活状态HSC减少主要通过HSC的凋亡来实现,抑制HSC激     

桂莪术提取物对人肝星状细胞的影响

目的：观察桂莪术提取物对肝星状细胞株（HSC-LX2）增殖、胶原生成以及基质金属蛋白酶表达的影响。方法：HSC-LX2 培养于含10%胎牛血清的DMEM 培养液中,用终浓度分别为0、22.5、45、90μmol·L-1 的桂莪术提取物处理24 h 后,MTT 法测定细胞增殖抑制率;LDH 比色法检测桂莪术提取物对HSC-LX2 的细胞毒性;ELISA 法检测HSC-LX2 I 型胶原表达的影响;Western Bolt 法检测HSC-LX2 基质金属蛋白酶-1（MMP-1） 蛋白表达。结果：MTT 结果显示,22.5、45、90 μmol·L-1 的桂莪术提取物均能抑制HSC-LX2 细胞增殖,呈剂量依赖性,抑制率分别为16.2%,43.9%,59.4%,IC50 为59.1 μmol·L-1;桂莪术提取物各剂量组培养上清液中LDH 活力差异无显著性;45、90 μmol·L-1 桂莪术提取物可显著降低I 型胶原的表达（P＜0.01）;Western Blot 结果分析显示,45、90 μmol·L-1 桂莪术提取物可显著提高HSC-LX2 MMP-1蛋白的表达（P＜0.05）。结论：桂莪术提取物可能通过抑制HSC-LX2 增殖和I 型胶原合成,提高MMP-1 蛋白表达,发挥抗肝纤维化作用。     

壮肝逐瘀煎对肝纤维化大鼠肝星状细胞凋亡的影响

目的:观察壮肝逐瘀煎对肝纤维化大鼠肝组织中肝星状细胞凋亡的影响,从细胞凋亡的角度探讨本方治疗肝纤维化的作用机理.方法:将100只Wistar大鼠随机分为正常对照组20只、观察组80只,参照相关文献复制大鼠肝纤维化模型,造模过程中,于第4周末随机处死正常对照组及观察组大鼠各5只,证实肝纤维化形成后,将观察组其余大鼠随机分为模型组、壮肝逐瘀煎大、中、小剂量组及秋水仙碱对照组,分别予生理盐水、壮肝逐瘀煎大、中、小剂量及秋水仙碱灌胃,日1次;12周后处死大鼠,观察肝脏组织病理学变化,采用免疫组化TUNEL、a-SMA 双标记检测肝星状细胞的凋亡情况.结果:壮肝逐瘀煎能加速活化肝星状细胞的凋亡,改善肝组织的纤维化程度.结论:壮肝逐瘀煎能有效抗大鼠肝纤维化,其作用机制是通过诱导星状细胞凋亡来实现的.     

阿米洛利对肝星状细胞增殖及分泌细胞外基质的影响

阿米洛利(amilofide)为Na /H 交换泵(NHE)阻滞剂,是一种临床常用的保钾利尿药,近年来发现NHE与细胞的增殖、分化和凋亡密切相关.本实验应用HSC-T6细胞系,观察阿米洛利对HSC增殖及细胞外基质分泌的影响,以探索临床抗肝纤维化的新途径.     

垂盆草总黄酮影响肝星状细胞凋亡的作用机制研究

目的:研究垂盆草总黄酮(SSTF)对大鼠肝星状细胞(HSC-T6)凋亡的影响及其作用机制。方法:将不同质量浓度的SSTF与HSC-T6细胞共培养不同时间后,MTT法检测SSTF对HSC-T6细胞的增殖抑制作用;流式细胞仪Annexin-V/PI双染方法检测SSTF对HSC-T6细胞凋亡的影响;应用Western blotting和Real-time PCR方法观察药物对凋亡相关细胞因子Bcl-2,Bax和Caspase-3蛋白及mRNA表达的影响。结果:SSTF能显著抑制HSC-T6细胞增殖、诱导细胞凋亡,且呈剂量和时间依赖;Western blotting结果显示,SSTF通过抑制Bcl-2,促进Bax和Caspase-3蛋白表达促进细胞凋亡;进一步采用Real-time PCR研究发现,Bcl-2能够从mRNA水平抑制Bcl-2、促进Bax和Caspase-3表达。结论:SSTF具有促进HSC-T6凋亡的作用,这种作用的发挥主要是通过抑制Bcl-2、促进Bax和Caspase-3蛋白和mRNA的表达实现的。     

苦参碱对肝星状细胞P75表达的影响

目的研究苦参碱对肝星状细胞P75表达的影响,探讨苦参碱促进肝星状细胞凋亡的可能机制。方法体外培养大鼠肝星状细胞系rHSC-99,用不同浓度的苦参碱（0.05 mg/mL、0.1 mg/mL、0.2 mg/mL、0.4 mg/mL）干预培养,采用免疫组化和半定量RT-PCR方法检测各浓度组大鼠肝星状细胞的P75 mRNA及蛋白的表达。结果苦参碱明显促进肝星状细胞（HSC）的P75 mRNA及蛋白的表达。结论苦参碱诱导HSC凋亡的机制,可能与其促进HSC的P75的表达有关。     

肝星状细胞的凋亡机制研究进展

大量研究发现激活状态的肝星状细胞(HSC)减少主要通过凋亡.而凋亡机制到目前为止仍然未清楚.其凋亡机制非常复杂,而很多的研究都提示HSC的凋亡与细胞因子及细胞外基质,NF-kB及其他因素有关.而控制细胞的生死程序以基因的形式存储于细胞中.当凋亡诱导因素作用细胞时按死亡程序凋亡.笔者就其机制作一综述.     

复方六月青诱导肝星状细胞凋亡的研究

目的:研究复方六月青(CLYQ)对肝星状细胞(HSC)凋亡的影响。方法:采用四甲基噻唑蓝(MTT)法检测CLYQ对细胞的毒性。采用吖啶橙荧光染色和流式细胞术检测CLYQ对HSC凋亡的影响。结果:CLYQ对HSC毒性很低。吖啶橙荧光染色法结果显示,与空白对照组相比较,CLYQ对HSC-T6凋亡细胞比例的影响有统计学差异(P<0.01),其凋亡率细胞比例有明显的剂量依赖性。流式细胞术结果显示,空白对照组凋亡细胞比例为4.82,CLYQ 3.6mg/mL浓度组凋亡细胞比例为10.10,1.8mg/mL浓度组凋亡细胞比例为6.76,0.9mg/mL浓度组凋亡细胞比例为5.18。结论:CLYQ通过诱导HSC细胞凋亡,从而发挥抗肝纤维化作用。     

Cortex Dictamni extract induces apoptosis of activated hepatic stellate cells via STAT1 and attenuates liver fibrosis in mice

Ethnopharmacological relevance

In traditional Chinese medicines, Cortex Dictamni is prescribed for the treatment of a variety of inflammatory diseases such as acute rheumatoid arthritis, skin inflammation and jaundice.

Aim of the study

This study was designed to investigate the effect of ethanol extract of Cortex Dictamni on treatment of hepatic fibrosis and its possible mechanisms.

Materials and methods

The in vivo effect of Cortex Dictamni extract (CDE) was evaluated by measuring histological changes and collagen content in CCl₄-indcued hepatic fibrosis mice. Viability, apoptosis and protein expression of hepatic stellate cells (HSC) were analyzed by MTT, Annexin V staining and Western blot respectively.

Results

CDE alleviated CCl₄-induced hepatic fibrosis in mice and showed a much stronger inhibition of cell viability in activated HSC cell line HSC-T6 than that in normal hepatocyte L02 cells. Furthermore, CDE induced apoptosis of HSC-T6 cells associated with increased expressions of cleaved PARP and cleaved caspase-3. Interestingly, CDE activated STAT1 in HSC-T6 cells and the effect of CDE on apoptosis of HSC-T6 cells could be neutralized using JAK/STAT1 signaling inhibitor AG490.

Conclusions

These findings suggest that CDE possesses anti-fibrosis activity with selectively induction of activated HSC apoptosis via activating STAT1, which might be a novel strategy for hepatic fibrosis therapy.     

Tetrandrine induces apoptosis in hepatic stellate cells

We have previously reported that tetrandrine reduced hepatic stellate cell activation and collagen accumulation in liver fibrosis induced by biliary obstruction. In the present study, we examined the apoptosis-inducing effect of tetrandrine on activated hepatic stellate cells, as the therapeutic goal in hepatic fibrosis is to eliminate the activated hepatic stellate cells by apoptosis. We used rat hepatic stellate cells transformed by Simian virus 40 (T-HSC/Cl-6) to overcome the limitations inherent in studying primary cultures of hepatic stellate cells. Tetrandrine treatment at doses of 25 and 50 microg/ml for 12 h induced apoptosis as confirmed by DNA fragmentation and increased sub-G1 DNA content as detected by flow cytometric analysis. Tetrandrine also induced the activation of caspase-3 protease and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results demonstrate that tetrandrine induces apoptosis of T-HSC/Cl-6 cells, and these results should contribute to the development of new agents for the treatment of hepatic fibrosis.     

Icaritin induces cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorates the development of liver fibrosis in rats

Aim of the study

Icaritin is an active ingredient extracted from the plant Herba Epimedium Sagittatum (Sieb. et Zucc.) Maxim. The purpose of this study is to investigate the effects and mechanisms of icaritin-induced cell death in activated hepatic stellate cells (HSCs) and ameliorating the development of liver fibrosis in rats.

Materials and methods

: In vitro, icaritin-induced cell death rates in HSC-T6 (rat) and LX-2 (human) HSC lines as well as normal hepatocyte cell lines HL-7702 (L02) and WRL-68 were assayed by MTT method, and the apoptotic ratios were detected by both flow cytometry and the Annexin-V-FITC Apoptosis Detection Kit. A Whole Rat Genome Microarray Kit was used to identify expression of interest genes through fold-change screening. In vivo study, experimental liver fibrosis models were built by carbon tetrachloride (CCl₄) or common bile duct ligation (CBDL) in Wistar rats. Icaritin (1 mg/kg/day, three days a week) was administered by gastric gavage for four weeks (n = 6 per group). At the end of the study, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) as well as the contents of hydroxyproline and collagen I in liver tissues were measured. Histopathological changes and the distribution of activated HSCs were observed in the liver tissues using hematoxyline-eosin (HE) staining and immunohistochemical staining for α-smooth muscle actin (α-SMA).

Results

Icaritin induced apoptosis in HSC-T6 and LX-2 in a concentration- and time-dependent manner with little toxicity to normal hepatocyte cell lines. The IC₅₀ of icaritin in HSC-T6 was 12.83 μM at 48 h. Apoptotic ratio of HSC-T6 treated with 24 μM icaritin was 20.19%, and the G2 phase of the cell cycle did not occur (P < 0.05). Gene analysis showed that icaritin up-regulated Bak-1, Bmf and Bax expression while significantly down-regulated Bcl-2 expression (vs. control group, P < 0.01). These results suggested that mitochondrial pathway played an important role in icaritin-induced apoptosis in activated HSCs. In vivo results showed that icaritin reduced the number of activated HSCs, and brought the elevated levels of AST, ALT, hydroxyproline and collagen I to normal or near normal values (vs. model group, P < 0.05).

Conclusions

Icaritin can induce cell death in activated HSCs through mitochondria-mediated apoptosis, ameliorate the progression of hepatic fibrosis in rats, and could be a promising drug for treating liver fibrosis.     

Study on antifibrotic effects of curcumin in rat hepatic stellate cells

Suppression of activation or fibrogenesis and induction of apoptosis, in hepatic stellate cells (HSCs) have been proposed as therapeutic strategies against liver fibrosis. Curcumin, an active compound isolated from yellow curry pigment of turmeric (Curcuma longa Linn), has been demonstrated to be an effective anti‐inflammatory and antioxidant compound. In this study, we investigated the in vitro antifibrogenic effects of curcumin on HSCs at the concentration range of (1–40 µM). A cell line of rat HSCs (HSC‐T6) was stimulated with transforming growth factor‐β1 (TGF‐β1). The inhibitory effects of curcumin (1.25～10 µM) on fibrosis‐related markers including α‐smooth muscle actin (α‐SMA) and collagen were assessed. In addition, the induction effects of curcumin (20～40 µM) on apoptosis in HSC‐T6 cells were also assessed by Hoechst and propidium iodide stains. Curcumin (1.25～10 µM) concentration‐dependently suppressed TGF‐β1‐induced α‐SMA expression and collagen deposition in HSC‐T6 cells, without cytotoxicity. Whereas, higher concentrations of curcumin (20～40 µM) induced cell apoptosis and cytochrome c release in HSC‐T6 cells. Our results suggest that curcumin exerted antifibrotic effects, possibly through two different mechanisms depending on its concentrations. At lower concentrations (1.25～10 µM), curcumin exerted antifibrogenic effects, whereas at higher concentrations (20～40 µM), curcumin exerted induction of apoptosis in HSCs. Copyright © 2009 John Wiley & Sons, Ltd.     

Tetrandrine inhibits activation of rat hepatic stellate cells stimulated by transforming growth factor-beta in vitro via up-regulation of Smad 7

Tetrandrine is a bisbenzylisoquinoline alkaloid derived from the root of a Chinese herbal medicine Stephania tetrandra S. Moore, which has been used traditionally for the treatment of hepatofibrogenic disease in China for several decades. In the present study, the inhibitory effects of tetrandrine lower concentrations (0.25, 0.5, 1, 2 mg/L) on culture-activation and transforming growth factor-β₁ (TGF-β₁)-stimulated activation of quiescent rat hepatic stellate cells (HSCs) in vitro were assessed, and the possible relations between the underlying mechanism of these effects and TGF-β signaling via its receptors were investigated. As shown by the examination of -SMA using immunocytochemical staining or Western blot, tetrandrine inhibited both culture-activation and TGF-β₁-stimulated activation of HSCs. Further investigations revealed that, in this process, TGF-β₁ mRNA expression was suppressed significantly in contrast to an up-regulation of Smad 7, while the expressions of type I and type II TGF-β₁ receptors and Smad 3 mRNA were insignificantly changed by tetrandrine. These results suggest that tetrandrine at lower concentrations has a significant inhibiting effect on culture-activation and TGF-β₁-stimulated activation of rat HSCs, and that it may be due to an up-regulation of Smad 7 which in turn blocks TGF-β₁ expression and its downstream signaling.     

Suppressive effects of YiGanKang, a combination of Chinese herbs, on collagen synthesis in hepatic stellate cell

Background/Aim

Clinical practice and animal research demonstrated that YiGanKang, a combination of Chinese herbs, has anti-fibrosis effects in chronic liver diseases. However, the mechanism is not clear. The present study is to investigate the inhibiting mechanism of YiGanKang on collagen type I synthesis induced by Interleukin-1β(IL-1 β) in rat hepatic stellate cells (HSCs).

Methods

Cultured rat HSCs were divided into 4 groups, control, IL-1β treated group, IL-1β + YiGanKang group and IL-1β + SB203580 (the p38 mitogen-activated protein kinase inhibitor) treated group. The expression of p38 mitogen-activated protein kinase (p38 MAPK), was evaluated by Western blot, collagen type I synthesis was examined by ³H-Pro incorporation.

Results

Type I collagen synthesis in HSCs increased significantly under the stimulation of IL-1β for 24 h, YiGanKang could inhibit p38 expression and type I collagen synthesis, SB203580, a p38 inhibitor, can significantly reduce type I collagen synthesis.

Conclusion

IL-1β could stimulate the synthesis of type I collagen in rat HSCs, p38 mediate signal pathway between IL-1β and was type I collagen production. YiGanKang inhibits HSCs collagen synthesis induced by IL-1β via p38 inhibition.     

A Chinese herbal medicine, Gexia-Zhuyu Tang (GZT), prevents dimethylnitrosamine-induced liver fibrosis through inhibition of hepatic stellate cells proliferation

Ethnopharmacological evidence

Gexia-Zhuyu Tang (GZT), also called Gexiazhuyu decoction (GXZYD), is a traditional Chinese herbal medicine for chronic liver diseases such as cirrhosis and liver fibrosis.

Aim of the study

In this study, we have investigated the affects of GZT on a rat model of dimethylnitrosamine (DMN)-induced liver fibrosis.

Materials and methods

In this study, the protective effects of GZT on DMN-induced liver fibrosis were measured using a rat model. Following 5 weeks of DMN-treatment (8 mg/kg, i.p., given 3 consecutive days each week), oral administration of GZT at 1.8 g/kg daily via oral gavage for 2 weeks beginning at week 13.

Results

Both body and liver weights were significantly decreased. The reductions in body and liver weights corresponded with increasing liver damage severity. Furthermore, GZT-treatment remarkably decreased the levels of serum GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase), and the mRNA expression levels of collagen alpha-1(I) and alpha-smooth muscle actin (alpha-SMA) in DMN-induced hepatic fibrosis. In addition, hepatic stellate cells (HSCs) play a major role in various types of liver fibrosis through initial myofibroblast transformation. The proliferation of HSCs was inhibited by GZT. Treatment with GZT also induced HSC apoptosis in a dose- and time-dependent manner. GZT treatment induced HSC apoptosis by facilitating Ca²⁺ release from the mitochondria within 6 h. Subsequently, caspases 3 and 12 were elevated by 72 h after treatment.

Conclusions

Our studies indicate that GZT exhibited both hepatoprotective and antifibrogenic effects in DMN-induced hepatic injury. These findings suggest that GZT may be useful in preventing the development of hepatic fibrosis.     

乙肝散对肝星状细胞增殖及凋亡的影响

目的通过观察中药乙肝散的药物血清对肝星状细胞(HSC/T6)的增殖及凋亡的影响,从细胞学和分子生物学水平探讨中药乙肝散的抗纤维化的作用机制,为临床用药提供理论依据。方法①MMT法测定细胞增殖。②用流式细胞仪测定各组各浓度药物血清对HSC/T6作用后的DNA含量,低于G1期DNA含量主峰(亚G1期峰)的细胞为凋亡细胞。结果5%,10%和20%浓度的乙肝散的药物血清均对HSC/T6的增殖有显著的抑制作用,对HSC/T6的凋亡率分别为8.63%,14.56%和21.95%。结论中药乙肝散能抑制HSC/T6的增殖,诱导HSC/T6的凋亡,因此促进活化的HSC的凋亡可能是乙肝散抗肝纤维化的细胞分子学重要机制之一。     

针刺对四氯化碳致肝纤维化大鼠肝脏中细胞外基质生成的抑制作用

目的:观察针刺治疗对四氯化碳致大鼠实验性肝纤维化的改善作用以及对纤维化肝脏中细胞外基质主要成分表达的影响。方法:将46只大鼠随机分为正常组(10只)、模型组(12只)、非穴组(12只)、穴位组(12只)。采用50%CCl4橄榄油溶液腹腔注射进行肝纤维化造模,同时针刺穴位组大鼠的"太冲"期门"肝俞"并电针"足三里"进行治疗。非穴组取穴为上述各穴左侧平移0.5cm处。造模和治疗6周后,颈总动脉取血用酶联免疫吸附法检测血清透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PCⅢ)水平;然后处死大鼠,分离肝组织进行病理切片观察;并分离各组大鼠的肝星状细胞,以Western blot法检测α平滑肌肌动蛋白(α-SMA)、α1(Ⅰ)型胶原蛋白[α1(I)collagen]、纤连蛋白(fibronectin)、基质金属蛋白酶-9(MMP-9)及金属蛋白酶组织抑制剂-1(TIMP-1)的蛋白表达,以Real-time PCR法检测α-SMA、α1(I)collagen和fi-bronectin的mRNA表达。结果:与正常组相比,模型组大鼠各项肝纤维化血清指标(HA、LN、PCⅢ含量)均明显升高(P<0.01,P<0.05);与模型组相比,穴位组血清HA、LN水平显著下降(P<0.05),非穴组无明显降低。针刺穴位可以降低肝星状细胞中α-SMA、α1(I)collagen和fibronectin的蛋白与基因表达(P<0.01,P<0.05),并上调MMP-9的蛋白表达(P<0.05),而对TIMP-1无明显影响(P>0.05)。结论:针刺穴位能有效改善四氯化碳导致的大鼠肝纤维化损伤,减少肝星状细胞分泌细胞外基质,从而抑制肝纤维化。